Aptamers are nucleic acid molecules having specific binding affinity to molecules through interactions other than classic Watson-Crick base pairing.
Aptamers, like peptides generated by phage display or monoclonal antibodies (MAbs), are capable of specifically binding to selected targets and, through binding, block their targets' ability to function. Created by an in vitro selection process from pools of random sequence oligonucleotides (FIG. 1), aptamers have been generated for over 100 proteins including growth factors, transcription factors, enzymes, immunoglobulins, and receptors. A typical aptamer is 10-15 kDa in size (30-45 nucleotides), binds its target with sub-nanomolar affinity, and discriminates against closely related targets (e.g., will typically not bind other proteins from the same gene family). A series of structural studies have shown that aptamers are capable of using the same types of binding interactions (hydrogen bonding, electrostatic complementarity, hydrophobic contacts, steric exclusion, etc.) that drive affinity and specificity in antibody-antigen complexes.
Aptamers have a number of desirable characteristics for use as therapeutics including high specificity and affinity, biological efficacy, and excellent pharmacokinetic properties. In addition, they offer specific competitive advantages over antibodies and other protein biologics, for example:
1) Speed and control. Aptamers are produced by an entirely in vitro process. In vitro selection allows the specificity and affinity of the aptamer to be tightly controlled and allows the generation of leads against both toxic and non-immunogenic targets.
2) Toxicity and Immunogenicity. Aptamers as a class have demonstrated little or no toxicity or immunogenicity. In chronic dosing of rats or woodchucks with high levels of aptamer (10 mg/kg daily for 90 days), no toxicity is observed by any clinical, cellular, or biochemical measure. Whereas the efficacy of many monoclonal antibodies can be severely limited by immune response to antibodies themselves, it is extremely difficult to elicit antibodies to aptamers (most likely because aptamers cannot be presented by T-cells via the MHC and the immune response is generally trained not to recognize nucleic acid fragments).
3) Administration. Whereas all currently approved antibody therapeutics are administered by intravenous infusion (typically over 2-4 hours), aptamers can be administered by subcutaneous injection. This difference is primarily due to the comparatively low solubility and thus large volumes necessary for most therapeutic MAbs. With good solubility (>150 mg/ml) and comparatively low molecular weight (aptamer: 10-50 KD; antibody: 150 KD), a weekly dose of aptamer may be delivered by injection in a volume of less than 0.5 ml. Aptamer bioavailability via subcutaneous administration is >80% in monkey studies (Tucker, 1999).
4) Scalability and cost. Aptamers are chemically synthesized and consequently can be readily scaled as needed to meet production demand. Whereas difficulties in scaling production are currently limiting the availability of some biologics (e.g., Ebrel, Remicade) and the capital cost of a large-scale protein production plant is enormous (e.g., $500 MM, Immunex), a single large-scale synthesizer can produce upwards of 100 kg oligonucleotide per year and requires a relatively modest initial investment (e.g., <$10 MM, Avecia). The current cost of goods for aptamer synthesis at the kilogram scale is estimated at $500/g, comparable to that for highly optimized antibodies. Continuing improvements in process development are expected to lower the cost of goods to <$100/g in five years.
5) Stability. Aptamers are chemically robust. They are intrinsically adapted to regain activity following exposure to heat, denaturants, etc. and can be stored for extended periods (>1 yr) at room temperature as lyophilized powders. In contrast, antibodies must be stored refrigerated.
Diabetes Therapeutics. Diabetes is a disease involving abnormal regulation of glucose in the bloodstream. The insulin receptor (IR) is a surface receptor and is a tetramer of 2 alpha and 2 transmembrane beta chains linked by disulfide bonds. The insulin receptor, which is activated by insulin, is a tyrosine kinase receptor. Its activation leads to an increase in the storage of glucose with a concomitant decrease in hepatic glucose release to the circulation. The insulin receptor induces a cellular response by phosphorylating proteins on their tyrosine residues. The IR is known to phosphorylate several proteins in the cytoplasm, including insulin receptor substrates (IRSs) and Shc. Phosphatidylinositol 3-kinase (PKI3) is one signaling molecule that is activated by binding IRSs and is important in coupling the IR to glucose uptake. PKI3 mediates glucose uptake by the IR as well as a variety of other cellular responses by generating PI(3,4)P2 and PI(3,4,5)P3. PI(3,4)P2 and PI(3,4,5)P3 then function directly as second messengers to activate downstream signaling molecules by binding pleckstrin homology (PH) domains in these signaling molecules.
The major function of insulin is to counter the concerted action of a number of hyperglycemia-generating hormones and to maintain low blood glucose levels. Because there are numerous hyperglycemic hormones, untreated disorders associated with insulin generally lead to severe hyperglycemia and shortened life span. Insulin is synthesized as a preprohormone in the b cells of the islets of Langerhans. Its signal peptide is removed in the cisternae of the endoplasmic reticulum and it is packaged into secretory vesicles in the Golgi, folded to its native structure, and locked in this conformation by the formation of 2 disulfide bonds. Specific protease activity cleaves the center third of the molecule, which dissociates as C peptide, leaving the amino terminal B peptide disulfide bonded to the carboxy terminal A peptide. Insulin secretion from b cells is principally regulated by plasma glucose levels, but the precise mechanism by which the glucose signal is transduced remains unclear. One possibility is that the increased uptake of glucose by pancreatic b-cells leads to a concomitant increase in metabolism. The increase in metabolism leads to an elevation in the ATP/ADP ratio. This in turn leads to an inhibition of an ATP-sensitive K+ channel. The net result is a depolarization of the cell leading to Ca2+ influx and insulin secretion. Chronic increases in numerous other hormones—including growth hormone, placental lactogen, estrogens, and progestins—up-regulate insulin secretion, probably by increasing the preproinsulin mRNA and enzymes involved in processing the increased preprohormone. In contrast, epinephrine diminishes insulin secretion by a cAMP-coupled regulatory path. In addition, epinephrine counters the effect of insulin in liver and peripheral tissue, where it binds to b-adrenergic receptors, induces adenylate cycles activity, increases cAMP, and activates PKA activates PKA similarly to that of glucagon. The latter events induce glycogenolysis and gluconeogenesis, both of which are hyperglycemic and which thus counter insulin's effect on blood glucose levels. In addition, epinephrine influences glucose homeostasis through interaction with a-adrenergic receptors. Insulin secreted by the pancreas is directly infused via the portal vein to the liver, where it exerts profound metabolic effects. These effects are the response of the activation of the insulin receptor which belongs to the class of cell surface receptors that exhibit intrinsic tyrosine kinase activity. With respect to hepatic glucose homeostasis, the effects of insulin receptor activation are specific phosphorylation events that lead to an increase in the storage of glucose with a concomitant decrease in hepatic glucose release to the circulation.
In most other tissues insulin increases the number of plasma membrane glucose transporters, but in liver glucose uptake is dramatically increased because of increased activity of the enzymes glucokinase, phosphofructokinase-1 (PFK-1), and pyruvate kinase (PK), the key regulatory enzymes of glycolysis. The latter effects are induced by insulin-dependent activation of phosphodiesterase, with decreased PKA activity and diminished phosphorylation of pyruvate kinase and phosphofructokinase-2, PFK-2. Dephosphorylation of pyruvate kinase increases its' activity while dephosphorylation of PFK-2 renders it active as a kinase. The kinase activity of PFK-2 converts fructose-6-phosphate into fructose-2,6-bisphosphate (F2,6BP). F2,6BP is a potent allosteric activator of the rate limiting enzyme of glycolysis, PFK-1, and an inhibitor of the gluconeogenic enzyme, fructose-1,6-bisphosphatase. In addition, phophatases specific for the phosphorylated forms of the glycolytic enzymes increase in activity under the influence of insulin. All these events lead to conversion of the glycolytic enzymes to their active forms and consequently a significant increase in glycolysis. In addition, glucose-6-phosphatase activity is down-regulated. The net effect is an increase in the content of hepatocyte glucose and its phosphorylated derivatives, with diminished blood glucose. In addition to the latter events, diminished cAMP and elevated phosphatase activity combine to convert glycogen phosphorylase to its inactive form and glycogen synthase to its active form, with the result that not only is glucose funneled to glycolytic products, but glycogen content is increased as well.
Insulin therapy is the only treatment for Type 1 diabetic patients. Occasionally, Type 2 diabetic patients are also treated with insulin. Type 2 diabetic patients usually require larger doses of insulin to achieve the target blood glucose value. At present, two methods of insulin delivery are available in the USA; multiple daily insulin injections and an insulin pump. Nasal insulin therapy is currently undergoing clinical trials and is not yet approved by the FDA for general use. All insulins sold in the United States today are of U-100 strength, 100 units of insulin per cc of fluid. There are other dilutions in other countries. Dosing is at least three times a day with meals.
Insulin generates its intracellular effects by binding to a plasma membrane receptor, which is the same in all cells. The receptor is a disulfide-bonded glycoprotein. One function of insulin (aside from its role in signal transduction.) is to increase glucose transport in extrahepatic tissue is by increasing the number of glucose transport molecules in the plasma membrane. Glucose transporters are in a continuous state of turnover. Increases in the plasma membrane content of transporters stem from an increase in the rate of recruitment of new transporters into the plasma membrane, deriving from a special pool of preformed transporters localized in the cytoplasm. In addition to its role in regulating glucose metabolism, insulin stimulates lipogenesis, diminishes lipolysis, and increases amino acid transport into cells. Insulin also modulates transcription, altering the cell content of numerous mRNAs. It stimulates growth, DNA synthesis, and cell replication, effects that it holds in common with the IGFs and relaxin.
The most common method of insulin delivery is subcutaneous injection. Another method is an insulin pump. The biggest advantage of an insulin pump is greater flexibility in the timing of meals, the patient does not have to eat at a particular time as is the case with insulin injection therapy. Meals can be skipped without the fear of low blood sugar. The disadvantages of insulin pump delivery are the risk of skin infection at the needle site, insulin delivery can be halted due to mechanical problems which can result in severe hyperglycemia (high blood glucose) and even diabetic ketoacidosis (a life-threatening condition), and cosmetic problems.
Despite the benefits of insulin therapy to treat type 1 diabetes, there are difficulties with regulation of effective plasma levels of insulin therapeutics. There is therefore a need for a therapeutic that effectively regulates insulin therapeutics in vivo.